The goal of this research is to establish a conceptual framework for the physiological role of lipolytic enzymes in intestinal lipid digestion, cholesterol absorption, and transport. The hypothesis is that the lipolytic enzymes modulate the structure and composition of cholesterol carriers in the intestinal lumen and the lipid composition of intestinal brush border membranes, thereby influencing cholesterol partitioning between the two compartments and dictating cholesterol absorption efficiency and chylomicron production. Our earlier data have shown that carboxyl ester lipase (CEL) influences chylomicron production, possibly by a mechanism related to its ability to hydrolyze ceramides in the intestinal tract. Our results also showed that pancreatic triglyceride lipase (PTL) gene deletion only delays but does not affect total amount of triglyceride absorption, but significantly decreases both the rate and the amount of cholesterol absorbed. This application plans to identify the mechanisms for each of these processes. Aim 1 will generate transgenic mice expressing mutagenized CEL with only esterolytic or lipoamidase activity in CEL-null background to test the hypothesis that the esterolytic activity of CEL is required for digestion and absorption of cholesteryl esters and complements PTL in fat digestion, whereas the lipoamidase activity of CEL is required for chylomicron assembly because of its ability to hydrolyze ceramides. Aim 2 will generate double knockout mice to test the effectiveness of CEL versus PTL related protein 2 (PTLRP2) as compensatory enzyme for PTL in fat digestion. Aim 3 will identify the mechanism by which PTL deficiency delays fat absorption and inhibits cholesterol absorption, testing the hypothesis that complete triglyceride digestion in the proximal intestine is necessary for cholesterol transfer to bile salt micelles prior to its absorption by the enterocytes, and that the reduced bile salt levels in the distal intestinal lumen significantly inhibit this process. We will also test the alternative hypotheses that reduced cholesterol absorption efficiency in distal intestine is related to the lack of expression of the putative cholesterol transporter NPC1L1 in distal intestine. These studies will identify lumenal events that are important for cholesterol absorption, and the mechanism that governs the difference in cholesterol absorption efficiency between proximal and distal intestine. These results will facilitate design of therapeutic strategy to lower cholesterol absorption and reduce the risk of coronary heart disease.